Novel gene nedl-1

ABSTRACT

Diagnostic agent or kit for the prognosis of neuroblastoma containing a nucleic acid probe or primer utilizing the nucleic acids derived from the NEDL-1 gene or the NEDL-1 protein, as well as method for diagnosing the prognosis of neuroblastoma.

TECHNICAL FIELD

This invention relates to nucleic acids derived from genes expressed inneuroblastoma and gene expression products encoded by the nucleic acids.More particularly, the invention relates to nucleic acids and theirfragments derived from the marker genes whose expression is enhanced inneuroblastomas with favorable prognosis based on comparison betweenneuroblastomas with favorable prognosis and neuroblastomas withunfavorable prognosis as well as to their utility in the diagnosis forthe prognosis of neuroblastomas.

BACKGROUND ART

(Tumorgenesis and Genes)

Individual tumors exhibit distinct characteristic natures, and theirbiological properties are not necessarily identical even though thebasic principle of oncogenesis is the same. Rapid advances in theunderstanding of cancer from a molecular biological and moleculargenetic perspective in recent years have opened the way to anexplanation of oncogenesis and tumor cell biology on the genetic level.

(Neuroblastomas)

Neuroblastoma is a pediatric cancer occurring in sympatheticgangliocytes and adrenal medullary cells which originate from cells ofthe peripheral sympathetic nervous system. Of these sympathetic nervoussystem cells, neural crest cells in the initial stage of developmentmigrate to the abdomen, differentiating and maturing at sites wheresympathetic ganglia are formed. Some of these cells migrate further tothe adrenal bodies, penetrating through the adrenal cortex which isalready in the process of formation, and reaching the medulla andforming medullary substance there. The neural crest cells also serve asa source of other peripheral nerve cells, differentiating into dorsalroot ganglia (sensory nerves), skin pigment cells, thyroid C cells, somepulmonary cells, intestinal gangliocytes, and the like.

(Prognosis of Neuroblastoma)

Neuroblastoma is characterized by a varied clinical profile (Nakagawara,Shinkeigashu no Hassei to Sono Bunshi Kiko [Neuroblastoma Developmentand Molecular Mechanism], Shoni Naika 30, 143, 1998). For example,neuroblastoma occurring at less than one year of age has very favorableprognosis, with the majority undergoing differentiation and cell death,and spontaneous regression. Currently, most neuroblastomas discovered bya positive result in the commonly performed mass screening of6-month-old infant urine are of the type which tend to undergo thisspontaneous regression. On the other hand, neuroblastoma occurring atage 1 or higher is highly malignant and leads to death of the infant inthe majority of cases. It is also hypothesized that a somatic mutationoccurs in highly malignant neuroblastomas in infants older than one yearof age, which are of monoclonal nature, whereas in naturally regressingneuroblastomas, the genetic mutation remains at only a germlinemutation. See Knudson A G, et al.: Regression of neuroblastoma IV-S: Agenetic hypothesis, N. Engl. J. Med. 302, 1254 (1980)).

(Genes which Allow the Diagnosis for Prognosis of Neuroblastoma)

With recent advances in molecular biology research, it has become clearthat expression of the high affinity nerve growth factor (NGF) receptorTrkA is closely connected with control of differentiation and celldeath. See Nakagawara A., The NGF story and neuroblastoma, Med. Pediatr.Oncol., 31, 113 (1998). Trk is a membrane-spanning receptor, existing asthe three main types, Trk-A, -B and -C.

These Trk family receptors play an important role in specific nerve celldifferentiation and survival in the central nervous and peripheralnervous systems. See Nakagawara, et al., Shinkeigasaiboushu ni OkeruNeurotrophin Juyoutai no Hatsugen to Yogo [Expression of NeurotrophinReceptors and Prognosis in Neuroblastoma], Shoni Geka (PediatricSurgery), 29: 425-432, 1997. The survival and differentiation of tumorcells is controlled by signals from Trk tyrosine kinase and Ret tyrosinekinase. In particular, the role of TrkA receptor is most significant,with TrkA expression being notably high in neuroblastomas with favorableprognosis, and its signals exerting a powerful control over survival anddifferentiation of tumor cells, and cell death (apoptosis). Inneuroblastomas with unfavorable prognosis, on the other hand, TrkAexpression is significantly suppressed, while tumor development is aidedby a mechanism in which survival is promoted by signals from TrkB andRet.

It has become clear that amplification of the neural oncogene N-myc hasbecome clearly associated with the prognosis of neuroblastoma. SeeNakagawara, Nou-shinkeishuyo no Tadankai Hatsugan [MultistageOncogenesis of Cerebral and Neural Tumors], Molecular Medicine, 364, 366(1999). This gene, first cloned in neuroblastoma, is ordinarily onlypresent in a single copy per haploid set in normal cells andneuroblastomas with favorable prognosis, whereas it has been found to beamplified several dozen times in neuroblastomas with unfavorableprognosis.

Up till the present time, however, no oncogene other than N-myc is knownto be expressed in neuroblastomas, and absolutely no genetic informationother than that of N-myc has been known in relation to favorable orunfavorable prognosis.

DISCLOSURE OF THE INVENTION

This invention has been accomplished in light of these circumstances,and its object is to identify the base sequences of genes which arerelated to favorable or unfavorable prognosis of neuroblastoma, and toallow the diagnosis for the prognosis of neuroblastoma (whetherfavorable or unfavorable) based on their genetic information. Its objectis also to provide the information on the functions of proteins whichare the transcripts of the aforementioned genes.

As a result of conducting diligent research, the present inventorsexamined the prognoses of neuroblastomas and succeeded in constructingcDNA libraries from the respective clinical tissues with favorableprognosis and with unfavorable prognosis. Approximately 2400 clones wererespectively obtained from these two types of cDNA libraries and wereclassified according to the prognosis of neuroblastomas and carried outprofiling of the respective subsets.

Thus, the present inventors found that a group of genes showeddifferential expression levels among the abovementioned subsets andshowed enhanced expression levels only in clinical tissues ofneuroblastoma with favorable prognosis; one of the genes was designated“NEDL-1 (nblaOO78).” Moreover, the present inventors sequenced the wholelength of NEDL-1 gene, and conducted the functional analysis of NEDL-1protein encoded by the gene: the protein was found to be a ubiquitinligase of the HECT type.

Based on this finding the present inventors have made it possible toprovide genetic information (base sequence data etc.) which allowed thedetection and cloning of genes whose expression is enhanced only in theclinical tissues of neuroblastoma with favorable prognosis. Furtherbased on the base sequence data, the present inventors made it possibleto provide methods of diagnosis for prognosis and diagnostic agentstherefor and thus completed this invention.

Specifically, this invention provides a nucleic acid probe comprisingnucleic acid (a) or nucleic acid (b) described below:

-   -   (a) a nucleic acid having a portion of a base sequence set forth        in SEQ ID NO:2 in the Sequence Listing or a base sequence        complementary thereto; or    -   (b) a nucleic acid capable of hybridizing to the nucleic acid        comprising a base sequence set forth in SEQ ID NO:2 in the        Sequence Listing, or having a base sequence complementary to        said base sequence.

Preferably, the nucleic acid is DNA in the nucleic acid probe describedabove.

Also preferably, the nucleic acid has a base length of at least 20 basesin the nucleic acid probe.

Further preferably, the base sequence set forth in SEQ ID NO:2 is itsfull-length in the nucleic acid probe.

This invention also provides a diagnostic agent for the prognosis ofneuroblastoma comprising the nucleic acid probe described above as theeffective ingredient.

This invention further provides a primer containing DNA (a) or DNA (b)as described below:

-   -   (a) DNA having a portion of a base sequence set forth in SEQ ID        NO:2 in the Sequence Listing or a base sequence complementary        thereto; or    -   (b) DNA capable of hybridizing to the DNA comprising a base        sequence set forth in SEQ ID NO:2 in the Sequence Listing, or        having a base sequence complementary to said base sequence.

This invention also provides a kit for the prognosis of neuroblastomacomprising the primer described above as the effective ingredient.

This invention further provides a method for diagnosing the prognosis ofneuroblastoma, the method comprising detecting the presence or absenceof a nucleic acid comprising a base sequence set forth in SEQ ID NO:2 inthe Sequence Listing in a clinical tissue sample of neuroblastoma.

This invention also provides a method for diagnosing the prognosis ofneuroblastoma, the method comprising detecting the presence or absenceof a protein comprising an amino acid sequence set forth in SEQ ID NO:1in the Sequence Listing in a clinical tissue sample of neuroblastoma.

This invention additionally provides a method for diagnosing theprognosis of neuroblastoma, the method comprising contacting with aclinical tissue sample of neuroblastoma, (a) a nucleic acid having aportion of a base sequence set forth in SEQ ID NO:2 in the SequenceListing or a base sequence complementary thereto or (b) a nucleic acidcapable of hybridizing to the nucleic acid comprising a base sequenceset forth in SEQ ID NO:2 in the Sequence Listing, or having a basesequence complementary to said base sequence; and analyzing theexpression of a protein comprising an amino acid sequence set forth inSEQ ID NO:1 in the Sequence Listing or a level thereof.

Accordingly, the nucleic acids and the proteins are derived from themarker genes whose expression is enhanced in neuroblastomas withfavorable prognosis based on comparison between neuroblastomas withfavorable prognosis and neuroblastomas with unfavorable prognosis. Theinformation on the sequences of the nucleic acids and proteins willcharacteristically enable the diagnosis for the prognosis ofneuroblastoma.

Further, this invention provides a polyubiqutination agent comprising asthe effective ingredient, a protein comprising a base sequence set forthin SEQ ID NO: 1 in the Sequence Listing.

In the polyubiquitination agent, the substrate to be ubiquitinated ispreferably β-amyloid precursor protein (βAPP), β-amyloid precursorprotein intracellular region (AICD) or a superoxide dismutase mutant(SOD1).

This invention also provides a composition for modulating β-amyloidprecursor protein (βAPP), the composition comprising an effective amountof a protein comprising an amino acid sequence set forth in SEQ ID NO:1in the Sequence Listing to modulate the expression, the production orthe formation of β-amyloid precursor protein in a cell.

This invention also provides a composition for modulating β-amyloidprecursor protein (βAPP), the composition comprising an effective amountof a nucleic acid comprising a base sequence set forth in SEQ ID NO:2 inthe Sequence Listing to modulate the expression, the production or theformation of β-amyloid precursor protein in a cell.

Further, this provides a method for modulating the expression, theproduction or the formation of β-amyloid precursor protein in a cell,the method comprising administering an effective amount of a proteincomprising an amino acid sequence set forth in SEQ ID NO:1 in theSequence Listing to modulate the expression, the production or theformation of β-amyloid precursor protein in a cell.

Still further, this provides a method for modulating the expression, theproduction or the formation of β-amyloid precursor protein in a cell,the method comprising administering an effective amount of a nucleicacid comprising a base sequence set forth in SEQ ID NO:2 in the SequenceListing to modulate the expression, the production or the formation ofβ-amyloid precursor protein in a cell.

This invention also provides a composition for modulating superoxidedismutase (SOD1) activity, the composition comprising an effectiveamount of the protein comprising an amino acid sequence set forth in SEQID NO:1 in the Sequence Listing to modulate the superoxide dismutase(SOD1) activity in a cell.

This invention also provides a composition for modulating superoxidedismutase (SOD1) activity, the composition comprising an effectiveamount of a nucleic acid comprising a base sequence set forth in SEQ IDNO:1 in the Sequence Listing to modulate the superoxide dismutase (SOD1)activity in a cell.

This invention also provides a method for modulating superoxidedismutase (SOD1) activity in a cell, the method comprising administeringan effective amount of a protein comprising an amino acid sequence setforth in SEQ ID NO:1 in the Sequence Listing to modulate the superoxidedismutase (SOD1) activity.

Further, this invention provides a method for modulating superoxidedismutase (SOD1) activity in a cell, the method comprising administeringan effective amount of a nucleic acid comprising a base sequence setforth in SEQ ID NO:2 in the Sequence Listing to modulate the superoxidedismutase (SOD1) activity.

In the composition for modulating superoxide dismutase (SOD1) activityas well as in the method for modulating superoxide dismutase (SOD1)activity in a cell, SOD1 is preferably a mutant type thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the protein structure ofubiquitin ligases of the HECT type showing that each has a HECT domainat its C-terminus, plural WW domains at its center and a C2 domain atits N-terminus.

FIG. 1B is an alignment diagram showing the homology analysis betweenthe amino acid sequence of NEDL-1 protein and the amino acid sequence ofNEDL-2 protein, where each of the domains is underlined or boxed andconservative amino acids are indicated by asterisks.

FIG. 2 is an electropherogram showing the results of determination ofthe expression levels of the NEDL-1 gene in clinical samples ofneuroblastomas with favorable prognosis and with unfavorable prognosisby semi-quantitative PCR.

FIG. 3A is a figure corresponding to an electropherogram showing theresults of determination of the expression levels of the NEDL-1 gene innormal human tissues by semi-quantitative PCR.

FIG. 3B is a figure corresponding to an electropherogram showing theresults of determination of the expression levels of the NEDL-1 gene invarious neuroblastoma cell lines by semi-quantitative PCR.

FIG. 4 is a figure representing autoradiography of different tissueexpression of the NEDL-1 gene in normal human tissues as analyzed byNorthern blot.

FIG. 5 is an immunoblotted electropherogram showing the ubiquitin ligaseactivity of the NEDL-1 protein.

FIG. 6A is a Western blot showing cellular localization of the NEDL-1gene (Cos7 cell).

FIG. 6B is a Western blot showing cellular localization of the NEDL-1gene (CHP134 cell).

FIG. 7 is an immunoblotted electropherogram showing the interactionbetween the NEDL-1 protein and ACID as obtained by immunoprecipitationwith anti-NEDL-1 antibody and detection with anti-FLAG antibody.

FIG. 8 is an immunoblotted electropherogram showing the interactionbetween the NEDL-1 protein and ACID as obtained by immunoprecipitationwith anti-FLAG antibody and detection with anti-NEDL-1 antibody.

FIG. 9A is an immunoblotted electropherogram showing the ubiquitinationof βAPP and ACID by the NEDL-1 protein as obtained byimmunoprecipitation with anti-HA antibody and detection withanti-ubiquitin antibody.

FIG. 9B is an immunoblotted electropherogram showing the ubiquitinationof FLAG-ACID by the NEDL-1 protein as obtained by immunoprecipitationwith anti-FLAG antibody and detection with anti-ubiquitin antibody.

FIG. 10 is an immunoblotted electropherogram showing the ubiquitinationof βAPP and ACID by the NEDL-1 protein as obtained byimmunoprecipitation with anti-HA antibody and detection with an antibodythat recognizes ACID.

FIG. 11 is an immunoblotted electropherogram showing the interactionbetween the NEDL-1 protein and SOD1 mutants as obtained byimmunoprecipitation with anti-NEDL-1 antibody and detection withanti-FLAG antibody.

FIG. 12 is an immunoblotted electropherogram showing the interactionbetween the NEDL-1 protein and SOD1 mutants as obtained byimmunoprecipitation with anti-FLAG antibody and detection withanti-NEDL-1 antibody.

FIG. 13 is an immunoblotted electropherogram showing the ubiquitinationof SOD1 and SOD1 mutants by the NEDL-1 protein.

BEST MODE FOR CARRYING OUT THE INVENTION

The nucleic acids (which will be referred to as “the nucleic acid ofthis invention”) derived from the gene which is highly expressed inneuroblastomas with favorable prognosis (which will be referred to as“the NEDL-1 gene of this invention” or simply as “the NEDL-1 gene”) andthe protein encoded by the gene will be described in detail by referringto the preferred embodiments of the invention.

As stated above, the nucleic acids of this invention are derived fromthe NEDL-1 gene of the invention and they make up the gene or areobtained from the gene by an in vivo or in vitro process. There are nolimitations on the base lengths of the nucleic acids and here they willbe referred to as the nucleic acids of the invention, which includenucleic acid fragments corresponding to parts of the gene. When the baselengths are short, they can be synthesized by chemical techniques. Theterm “nucleic acid(s)” as used in this specification refers to, forexample, DNA or RNA, or polynucleotides derived therefrom which areactive as DNA or RNA, and preferably refers to DNA and/or RNA. Theparticularly preferred nucleic acid has a base sequence that isidentical with the human cDNA sequence disclosed in this specificationor that is complementary to the sequence.

The term “hybridize under stringent conditions” as used in thisspecification means that two nucleic acid fragments hybridize to eachother under the hybridization conditions described by Sambrook, J. etal. in “Expression of cloned genes in E. coli”, Molecular Cloning: ALaboratory Manual (1989), Cold Spring Harbor Laboratory Press, New York,USA, 9.47-9.62 and 11.45-11.61.

More specifically, the “stringent conditions” refers to hybridization atapproximately 45° C., 6.0×SSC, followed by washing at 50° C., 2.0×SSC.The stringency may be selected by choosing a salt concentration in thewashing step from approximately 2.0×SSC, 50° C. as low stringency toapproximately 0.2×SSC, 50° C. as high stringency. Also, the temperaturein the washing step may be increased from room temperature, orapproximately 22° C. as low stringency conditions, to approximately 65°C. as high stringency conditions.

The term “nucleic acid(s)” as used in this specification refers to anisolated nucleic acid(s) and to a nucleic acid or a polynucleotidecontaining substantially no cellular substances or culture medium, ifprepared by recombinant DNA techniques, or containing substantially noprecursor chemical substances or other chemical substances, if preparedby chemical synthesis.

The term “favorable prognosis” as used in this specification refers to acondition of neuroblastoma in which the tumor is localized or has becomea regressing or benign sympathetic ganglion neoplasm, and is judged tohave low malignancy based on N-myc or other tumor markers (TrkA,chromosomal aberration). According to a preferred embodiment of theinvention, a favorable prognosis is a case of stage 1 or 2, with anonset age of less than one year and survival without recurrence for 5 ormore years after surgery, and with no noted amplification of N-myc inthe clinical tissue; however, there is no limitation to such specificcases. The term “unfavorable prognosis” as used in this specificationrefers to a condition of neuroblastoma in which progression of the tumorhas been observed, and it is judged to have high malignancy based onN-myc or other tumor markers. According to a preferred embodiment of theinvention, an unfavorable prognosis is a case of stage 4, with an onsetage of greater than one year, death within 3 years after surgery andnoted amplification of N-myc in the clinical tissue; however, there isno limitation to such specific cases.

Neuroblastoma is a tumor consisting of actual nerve cells, of which onlytwo types of tumor are known in humans, and analysis of the genesexpressed therein is expected to provide very useful knowledge forunderstanding the biology of nerve cells. Specifically, it is extremelydifficult, and practically impossible, to obtain site-specifichomogeneous tissue from the brain or peripheral nerves. On the otherhand, a neuroblastoma consists of an almost homogeneous nerve cellpopulation (though tumorized) derived from peripheral sympathetic nervecells, and thus offers the high possibility of obtaining homogeneousexpression of neuro-related genes. Furthermore, since neuroblastoma is atype of cancer, it will characteristically have many important genesexpressed in the immature stage of neurogenesis.

Clinically and biologically, neuroblastoma can be neatly classified intofavorable prognosis and unfavorable prognosis types. Cancer cells fromneuroblastoma with favorable prognosis are characterized by having avery slow rate of proliferation, with spontaneous regression beginningat some point. Findings to date have confirmed that nerve celldifferentiation and apoptosis (nerve cell death) occur in thespontaneous regression, and that the differentiation which occurs in thematuration stages of normal nerve cells and programmed cell death arephenomena very closely resembling each other. Consequently, it is highlyprobable that the analysis of genes expressed in such tumors will leadto obtaining important genetic information relating to nerve celldifferentiation and apoptosis.

NEDL-1 gene from which the useful genetic information can be obtainedand the NEDL-1 protein encoded by the gene are found in clinical tissuesof human neuroblastomas with favorable prognosis. These gene and proteinare provided with the characteristics described below.

The NEDL-1 gene of this invention is a gene having the full length of6,200 bases (coding region of 4,755 bases) and its base sequence isshown in SEQ ID NO:2 in the Sequence Listing. NEDL-1 protein encoded bythe gene comprises 1585 amino acids and its full length is shown in SEQID NO:1 in the Sequence Listing. The base sequence and the amino acidsequence have been registered with GeneBank (HYPERLINKhttp://www.ncbi.nlm.nih.gov.) as Accession No. AB048365.

The present inventors found that as a result of the structural andfunctional analysis of the NEDL-1 gene and the NEDL-1 protein, NEDL-1 isa ubiquitin ligase of the HECT type. FIG. 1 shows the results from thehomology analysis between the NEDL-1 protein and KIAA03222 protein(NEDL-2) which is a known member of the HECT type ubiquitin ligasefamily. The NEDL-1 protein has the domains characteristic of HECT typeubiquitin ligase. Specifically, these are (1) HECT domain (about 300amino acids) at the C-terminus, which is positions 1280-1585 in NEDL-1;(2) plural WWW domains at the central part (about 35 to 40 amino acids),which is positions 807-841 and positions 998-1030 in NEDL-1 andpositions 806-840 in NEDL-2; (3) C2 domain at the N-terminus binding tomembrane lipid in a Ca-dependent manner, which is positions 185-295 inNEDL-1 and positions 186-295 in NEDL-2. In addition, the NEDL-1 proteinwas found to possess ubiquitin ligase activity at the same level as doesNedd4 which is a HECT type ubiquitin ligase.

Because proteins are decomposed in the ubiquitin-protease system, thissystem is essential to an adequate cellular process. In brief, thesystem allows a number of ubiquitin molecules (Ub) to be bound to atarget protein (what is called “polyubiquitination or ubiquitination)and the ubiquitinated protein is decomposed by 26S proteasome. It hasbeen elucidated that the ubiquitination of proteins progresses throughthe catalytic action of a series of enzyme groups, that isubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2),and ubiquitin-ligating enzymes (ubiquitin ligases). See, for example, areview of Keiji Tanaka, “Ubiquitin and Proteasome” in ExperimentalMedicine, Vol. 18, No. 11, pp. 1452-1456 (2000) by Yodosha. Among thoseenzymes ubiquitin ligase (E3) receives Ub from E2-Ub and ligate this Ubto the target protein (substrate). Thus, ubiquitin ligase is thought tobe most heavily involved in the specificity with which a specificprotein is ubiquitinated.

It has been pointed out that the anomaly in the ubiquitin-proteasomesystem is related to many diseases (R. J. Mayer et al., Biochem. Biophs.Acta 1089: 141-157 (1991)). Recently, the relation betweenneurodegenerative diseases and the anomaly in ubiquitin metabolism hasattracted attention; and there has been a report that E6-AP, which isknown as a ubiquitin ligase, is one of the responsible genes forAngelman syndrome (Nobutomi Honda et al., “HECT type ubiquitin-ligatingenzymes: physiological functions and disease state” in ExperimentalMedicine, Vol. 18, No. 11, pp. 1483-1490 (2000) by Yodosha). The NEDL-1protein of this invention, which is one of HECT type ubiquitin ligases,is highly expressed in nerve tissues. It is, therefore, well anticipatedthat the protein uses as a substrate the product of a causative gene ofa neurodegenerative disease. These causative gene products are believedto be beta amyloid precursor protein (βAPP), preselinin protein (PS) andothers.

As will be described in the Examples, it was actually found that NEDL-1interacted with amyloid beta precursor intracellular domain (AICD) whichwas the coding region of amyloid precursor protein. It was furtherdetermined that this interaction resulted from ubiquitination of BAPPand ACID by NEDL-1.

Amyloid is a protein that deposits in cerebral blood vessels and senileplaques of an Alzheimer patient: it is comprised principally ofβ-protein with a molecular weight of 4 kDa and is produced when amyloidprecursor protein is cleaved by secrease. The fact that NEDL-1 directlyinteracts with ACID leads to the possibility that NEDL-1 regulates theproduction of βAPP (then βamyloid) directly or indirectly. It will be afinding that is extremely important to planning a strategy for Alzheimertreatment which targets lowered production of β-amyloid on a molecularlevel.

As will also be described in the Examples, it was also found that NEDL-1interacted with superoxide dismutase mutants (SOD1). It was furtherdetermined that this resulted from ubiquitination of the SOD1 mutants byNEDL-1.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative diseases withunfavorable prognosis that involves muscular atrophy due to thedegeneration or deciduation of motor neurons. Currently, familial ALS isseen in a frequency of 5-10% of the total ALS. The causative gene hasbeen identified as the Cu/Zn superoxide dismutase (SOD1) in some of thefamilies. SOD1 is an enzyme that inactivates superoxide dismutase whichis one type of active enzymes produced in a cell during an aerobicprocess. There is the possibility that its lowered level causesdegeneration of nerve cells; however the detail of mechanism is unknown.The cause for other general type of amyotrophic lateral sclerosis isalso unknown.

Presently, the virus theory, the poisoning theory, the nerve nutrientfactor depletion theory, the autoimmune theory, the excessive glutamatetheory, the free radical theory and others are proposed. However, thedecisive pathological mechanism by which only motor nerve degenerates inALS has not been elucidated. The aggregate hypothesis assumes thatmutant SOD1 forms an aggregate in a cell and exhibits cytotoxity; andthis will be becoming the most convincing one in recent years.

Thus far about 80 of SOD1 mutants have been reported. Intracellularsignal transduction remain unknown for any of these mutants. There hasbeen a report on interacting molecules for two types of SOD1 mutants(G85R/G93A) and only two types, lysyl-tRNA synthetase andtranslocon-associated protein delta, have been identified as a proteinfactor that only binds to those mutants and that does not bind to normalSOD1. (Kunst C B, Mezey E, Brownstein M J, Patterson D, “Mutations inSOD1 associated with amyotrophic lateral sclerosis cause novel proteininteractions” Nat. Genet. 1997 January; 15(1) 91-4.) The details havenot yet been elucidated. Thus, about 130 years have passed since thefirst report of ALS, but even now the situation may be that theelucidation of the intracellular signal transduction for cytotoxitywhich the mutant SOD1 has newly acquired. In consideration of thepresent circumstance described above, the results obtained from thisinvention are believed to provide very useful information for theelucidation of the mechanism of ALS crisis that has not been hithertoclarified.

The NEDL-1 protein has an amino acid sequence set forth in SEQ ID NO:1in the Sequence Listing but this invention also encompasses a proteinhaving an amino acid sequence comprising a deletion, a substitution, aninsertion or an addition of one or more amino acids in the amino acidsequence set forth in SEQ ID NO:1 in the Sequence Listing.

This invention also encompasses salts of the NEDL-1 protein and others.These salts are not particularly limited and, for example, preferred area sodium salt, a potassium salt, a magnesium salt, a lithium salt and anammonium salt.

Sugar chains are added to many proteins and the addition of a sugarchain may be adjusted by converting one or more amino acids. Therefore,this invention encompasses proteins the sugar chain addition of whichhas been adjusted in the amino acid sequence set forth in SEQ NO:1 inthe Sequence Listing.

This invention further encompasses a nucleic acid having a base sequenceencoding the NEDL-1 protein. The term “encoding a protein” as usedherein means that either of complementary double strands has a basesequence encoding the protein when DNA is double-stranded. The nucleicacids of this invention embrace a nucleic acid comprising a basesequence directly encoding the amino acid sequence set forth in SEQ IDNO:1 in the Sequence Listing and a nucleic acid comprising a basesequence complementary to said nucleic acid.

Further, the nucleic acid of the invention may be a nucleic acidhybridizing to the nucleic acid comprising a base sequence set forth inSEQ ID NO:2 under stringent conditions. The base sequence is notparticularly limited insofar as it satisfies this condition. Stillfurther, the nucleic acids of the invention encompass a nucleic acidcomprising a base sequence complementary to the nucleic acidhybridizable under the stringent conditions mentioned above.Specifically there is mentioned a nucleic acid comprising deletions,substitutions, insertions or additions in some bases of the nucleic acidcomprising a base sequence set forth in SEQ ID NO:2 or a nucleic acidcomplementary to said nucleic acid. As used herein, the deletion, thesubstitution, the insertion and the addition include not only a shortdeletion, substitution, insertion and addition with 1 to 10 bases, butalso a long deletion, substitution, insertion and addition with 10 to100 bases.

As a result of comparing levels of expression of the NED-1 geneaccording to this invention in clinical tissues from neuroblastomas withfavorable prognosis and with unfavorable prognosis, a highly significantdifference was found. That is, expression of this gene was enhanced inneuroblastomas with favorable prognosis. Thus, in addition to providingthe useful genetic information described above, the nucleic acidsequence set forth in SEQ ID NO:2 can also be utilized as data for tumormarkers to diagnose favorable or unfavorable prognosis of neuroblastoma,by detecting the nucleic acid (DNA or RNA) having that sequence. Theamino acid sequence in SEQ ID NO:1 can also be utilized as data fortumor markers to diagnose favorable or unfavorable prognosis ofneuroblastoma, by detecting the NEDL-1 protein based on the sequenceinformation.

Specifically, by using the NEDL-1 gene and the NEDL-1 protein accordingto this invention, the invention will make it possible to obtain variousgenetic information on or relating to neuroblastoma through thefollowing means.

(1) Probes for Use in Hybridization

According to one embodiment of this invention, the nucleic acid of theinvention can be used as a probe (i.e., the probe of this invention) forhybridization to detect the NEDL-1 gene expressed in neuroblastoma. Thenucleic acid according to this invention can also be used as probes forhybridization in order to determine gene expression in a variety oftumors and normal tissues, to identify the distribution of the geneexpression.

When the nucleic acid according to this invention is used as a probe forhybridization, there are no particular limitations on the actual methodof hybridization. As preferred methods there may be mentioned, forexample, Northern hybridization, Southern hybridization, colonyhybridization, dot hybridization, fluorescence in situ hybridization(FISH), in situ hybridization (ISH), DNA chip methods, and microarraymethods.

As one application example of the hybridization, the nucleic acidaccording to this invention can be used as a probe for Northernhybridization to measure the length of mRNA or to quantitatively detectthe expression of the NEDL-1 gene of this invention in a clinical tissuesample to be assayed.

As another application example, the nucleic acid according to thisinvention can be used as a probe for Southern hybridization to detectthe presence or absence of the DNA sequence in the genomic DNA of aclinical tissue sample to be assayed.

As still another application example, the nucleic acid according to thisinvention can also be used as a probe for fluorescence in situhybridization (FISH) to identify the location of the NEDL-1 gene of thisinvention on a chromosome.

As a further application example, the nucleic acid according to thisinvention can also be used as a probe for in situ hybridization (ISH) toidentify the tissue distribution of expression of the NEDL-1 gene ofthis invention.

When the nucleic acid according to this invention is used as a probe forhybridization, a base length of at least 20 is necessary; and among thenucleic acids according to this invention, a nucleic acid having 20 ormore contiguous bases is preferably used. More preferably, the nucleicacid having 40 or more bases is used and most preferably the nucleicacid having 60 or more bases is used. Further, the nucleic acid havingthe full-length of the base sequence set forth in SEQ ID NO:2 may beused.

Nucleic acid probe techniques are well known to one skilled in the art,and for example, conditions suitable for hybridization between a probeof specific length according to the invention and the targetpolynucleotide may be readily determined. In order to obtainhybridization conditions optimal to probes of varying lengths, Sambrooket al. “Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor (1989) may be followed for such manipulations which are wellknown to one skilled in the art.

The probe according to this invention may preferably be labeled for usein an easily detectable fashion. The detectable label may be any typeand any element or compound which can be detected either visually orusing devices. As commonly used detectable labels, there may bementioned radioactive isotopes, avidin and biotin and fluorescentsubstances (FITC or Rhodamins). The radioactive isotopes are ³²p, ¹⁴C,¹²⁵I, ³H, ³⁵S etc. Biotin-labeled nucleotides may be incorporated intoDNA or RNA by nick translation, or chemical or enzymatic means. Thebiotin-labeled probes are detected after hybridization using labelingmeans such as avidin/streptavidin, fluorescent labels, enzymes, goldcolloidal complexes or the like. The nucleic acid probe of thisinvention may also be labeled by binding with a protein. For thispurpose, a radioactive or fluorescent histone single-stranded bindingprotein may also be used. In this manner, a suitably labeled probeconstitutes a diagnostic agent for prognosis according to thisinvention.

(2) Primers for Use in PCR

For methods of detecting the NEDL-1 gene according to this inventionother than the hybridization, primers can be designed after any nucleicacid (DNA) sequence contained in the nucleic acid according to thisinvention and the polymerase chain reaction (PCR) method can be used.For example, RNA may be extracted from a clinical tissue sample to beassayed, and the gene expression can be semi-quantitatively measured byRT-PCR. This may be carried out by a method well known to one skilled inthe art. For example, “Molecular Cloning: A Laboratory Manual,” (T.Maniatis, Cold Spring Harbor Laboratory Press)-or Idenshibyo Nyumon[Introduction to Genetic Diseases] (Takahisa, S.: Nankodo Publishing)may be followed.

When the nucleic acid according to this invention (DNA) is used as a PCRprimer (i.e., the primer of the invention), a base length of 10 to 60 isnecessary; and among portions of the base sequences according to theinvention, the nucleic acid having 10 to 60 contiguous bases ispreferably used. More preferably, one having 15 to 30 bases is used.Generally, a primer sequence with a GC content of 40-60% is preferred.Also, there is preferably no difference in the Tm values of the twoprimers used for amplification. The primer has such base sequence thatthere is no annealing at the 3′ ends of the primers and no secondarystructure is formed in the primers.

(3) Gene Screening

The nucleic acid according to this invention can also be used to detectthe expression distribution of the NEDL-1 gene which is expressed invarious tissues or cells. This can be accomplished, for example, byusing the nucleic acid according to this invention as a probe forhybridization or as a primer for PCR.

The expression distribution of the gene can also be detected using a DNAchip, microarray or the like. That is, the nucleic acid according to theinvention may be directly attached to the chip or array. There is knowna method by which nucleic acids (DNA) are spotted to a substrate for thepurpose of attaching them to a chip or array by using a high precisiondispenser (for example, see U.S. Pat. No. 5,807,522). mRNA extractedfrom a clinical tissue sample may be labeled with a fluorescentsubstance or the like, hybridized thereto, and an analysis can be madeof the type of tissue cells with high expression of the gene. The DNAattached to the chip or the array may be the reaction product of PCRusing the nucleic acid or its fragment according to the invention. As analternative method, the nucleic acid fragment of this invention (DNAfragment) may be directly synthesized on a substrate to form a DNA chipor a DNA array (See, for example, U.S. Pat. No. 5,424,186).

(5) Methods of Diagnosing Tumor Prognosis and Tumor Markers to be UsedTherefor

As mentioned above, the NEDL-1 gene of this invention has its expressionenhanced in neuroblastomas with favorable prognosis. Therefore, thenucleic acid according to this invention can be used as a probe forhybridization, or as a primer for PCR to determine the presence orabsence of enhancement in the gene expression in a sample containing theclinical tissue taken from the subject, which enables the identificationof prognosis. The methods of detecting the gene include Northern blothybridization, in situ hybridization and RT-PCR, as mentioned aboveamong others.

When hybridization is employed, prognosis may be diagnosed as favorableif the amount of nucleic acid hybridizing to the probe is increased inthe sample. When RT-PCR is employed, mRNA is extracted from the sampleand reverse transcribed into DNA, amplification is performed using theaforementioned primer, and the gene expression is semi-quantitativelymeasured. The prognosis may be diagnosed as favorable if the geneexpression is then found to be enhanced. For the purpose of suchspecific diagnosis it is preferred to utilize a diagnosis kit containinga pair of such primers as essential components. In addition to theprimer components, the diagnosis kit also include known components suchas PCR buffer, detergent solution and enzymes.

(6) Antisense Oligonucleotides

According to another embodiment of this invention there are providedantisense oligonucleotides to the nucleic acids of the invention. Theantisense oligonucleotides are capable of hybridizing to the nucleicacids of the invention, and include antisense DNAs and antisense RNAs.Antisense DNA inhibits transcription of mRNA from DNA, while antisenseRNA inhibits translation of mRNA. These antisense oligonucleotides maybe synthesized using an automated synthesizer or by PCR using thenucleic acid of the invention as templates. The antisenseoligonucleotides also encompass antisense oligonucleotide derivativeshaving improved binding affinity for DNA or mRNA, tissue selectivity,cell permeability, nuclease resistance and intracellular stability.These derivatives may be synthesized using antisense technology known inthe art.

Antisense oligonucleotides having sequences complementary to thesequences near the translation initiation codon of the mRNA, those ofthe ribosome-binding site, and those of the capping site or the splicingsite are capable of inhibiting synthesis of the RNA and therefore willexhibit a particularly notable inhibitory effect on gene expression.This invention therefore encompasses such antisense oligonucleotides.

(7) Gene Therapy

According to a further embodiment of this invention, there are providednucleic acid sequences encoding the therapeutic genes to be used in genetherapy. Thus, the nucleic acid of the invention can be transferred intoa vector for use in gene transportation, whereby the transgene (i.e.,the NEDL-1 gene of the invention) can be expressed by an arbitraryexpression promoter and can be used in the gene therapy forneurodegenerative diseases, for example.

1. Vectors

The transferable viral vectors may be prepared from DNA viruses or RNAviruses. They may be any viral vector of an MoMLV vector, a herpes virusvector, an Adenovirus vector, an AAV vector, a HIV vector, a SIV vector,a Seidai virus vector and the like. One or more proteins among theconstituent protein group of a viral vector are substituted by theconstituent proteins of a different species of virus, or alternatively apart of the nucleic acid sequence constituting genetic information issubstituted by the nucleic acid sequence of a different species of virusto form a viral vector of the pseudo-type which can also be used in thisinvention. For example, there is mentioned a pseudo-type viral vectorwherein the Env protein (an envelop protein of HIV) is substituted bythe VSV-G protein (an envelop protein of vesicular stomatitis virus orVSV) (Naldini L., et al., Science 272, 263-1996). Further, viruseshaving a host spectrum other than human are usable as the viral vectorinsofar as they are efficacious. As for the vectors other than those ofviral origin, there may be used complexes of calcium phosphate andnucleic acid, ribosomes, cation-lipid complexes, Seidai virus liposomes,polymer carriers having polycation as the backbone main chain andothers. In addition, methods such as electroporation and gene guns maybe used as a gene transfer system.

2. Expression Promoters

As for the expression cassettes to be used for the therapeutic gene, anycassettes without any particular limitations may be used insofar as theycan cause genes to express in the target cells. One skilled in the artcan readily select such expression cassettes. Preferably, they areexpression cassettes capable of gene expression in the cells derivedfrom an animal, more preferably, expression cassettes capable of geneexpression in the cells derived from a mammal, and most preferablyexpression cassettes capable of gene expression in the cells derivedfrom a human. The gene promoters that can be used as expressioncassettes include: for example, virus-derived promoters from anAdenovirus, a cytomegalovirus, a human immunodeficiency virus, a simianvirus 40, a Rous sarcoma virus, a herpes simplex virus, a murineleukemia virus, a sinbis virus, a hepatitis type A virus, a hepatitistype B virus, a hepatitis type C virus, a papilloma virus, a human Tcell leukemia virus, an influenza virus, a Japanese encephalitis virus,a JC virus, parbovirus B19, a poliovirus, and the like; mammal-derivedpromoters such as albumin, SRα, a heat shock protein, and an elongationfactor; chimera type promoters such as a CAG promoter; and the promoterswhose expression can be induced by tetracyclines, steroids and the like.

(8) Drugs

According to a still further embodiment of this invention, there areprovided therapeutic proteins and peptides as drugs. As will beconsidered in practicing this invention, the NEDL-1 protein of theinvention and its partial peptide may be prepared according to theformulation method of choice and may be used through any desired routeof administration and at any desired dosage age in the treatment ofmalignant tumors or neurodegenerative diseases (e.g., Alzheimer disease)of different types, for example.

1. Preparation Method

The drug may be prepared as a recombinant viral vector containing atherapeutic gene that is designed for therapeutic purposes as describedabove. More specifically, a recombinant virus vector comprising theNEDL-1 gene may be prepared by dissolving it in an appropriate solventsuch as water, physiological saline or an isotonized buffer solution.Alternatively, the NEDL-1 protein produced by any desired method may bedissolved in an appropriate solvent such as water, physiological salineor an isotonized buffer solution to prepare the vector similarly. Here,polyethylene glycol, glucose, various amino acids, collagen, albumin orthe like may be then added as protective materials for the preparation.

2. Administration Method and Dosage

There are no particular limitations on the method of administrating thedrug mentioned above to the living body. For example, parentaladministration, including injection is preferably carried out. The uselevel of the drug varies depending on the method of use, the purpose ofuse, etc.; and one skilled in the art can easily select as appropriateand optimize it. In the case of injection, for example, the daily dosageis preferably administered at about 0.1 μg/kg to 1000 mg/kg per day, andmore preferably at about 1 μg/kg to 100 mg/kg per day.

(9) Antibodies, Antisense, Ribozymes and TFO

In accordance with a still another embodiment of this invention, anantibody to suppress the ubiquitin activity of the NEDL-1 protein of theinvention and base sequences, including antisense, ribozyme or TFO, tosuppress the expression of the NEDL-1 gene of the invention areprovided. As will be considered in practicing this invention, nucleicacids encoding antisenses, ribozymes and TFOs can be transferred into avector used as a gene carrier; the transgene can be expressed by anysuitable expression promoter and can be used, for example, to establisha primary culture cell line or to construct a cancer model animal.

(10) Genetically Modified Animals

In accordance with a yet another embodiment of this invention, a nucleicacid sequence to knock out the expression of the NEDL-1 gene of theinvention and a knockout animal (e.g., knockout mouse) are provided.There are provided a transgenic animal (e.g., transgenic mouse) wherethe gene has been forcedly expressed and a genetically modified animalhaving an introduced mutant gene obtained by introducing an arbitrarymutation (such as a point mutation or deletion) into the gene. Thisgenetically modified animal can be used to construct a model animal fora neurodegenerative disease, for example.

As described above, by utilizing the NEDL-1 gene or the NEDL-1 proteinaccording to this invention or the information obtainable therefrom, itwill be possible to detect the NEDL-1 gene in a clinical tissue sample,which then will allow the diagnosis of neuroblastoma whether favorableor unfavorable prognosis. Further, by utilizing the gene, the protein orthe information obtainable therefrom, it will be possible to designtumor markers that can be used in the diagnosis for prognosis and theaforementioned method.

This invention will now be explained in greater detail by way of theexamples; however, the technical scope of invention will not berestricted to those examples.

EXAMPLES Preparation Example 1 Construction of cDNA Library FromNeuroblastoma

1. Obtaining Samples

The clinical tissue samples of neuroblastoma were quasi-asepticallyfrozen immediately after surgical extraction and then preserved at −80°C.

2. Selecting Samples with Favorable Prognosis

Prognosis of the samples obtained in 1. above was carried out based onthe following criteria. Favorable prognosis Unfavorable prognosis Stage1 or 2 Stage 4 Age of onset: <1 Age of onset: ≧1 Survival for ≧5 yearsDeath within 3 years after surgery without after surgery recurrence Noamplification of N- Amplification of N-myc myc

Amplification of N-myc in the aforementioned two sample types wasconfirmed in the following manner.

The samples obtained in 1. above was thinly sliced with a scalpel andthen thoroughly homogenized after addition of 5 ml of TEN buffer (50 mMTris-HCl (pH=8.0)/1 mM EDTA/100 mM NaCl). Upon adding 750 μl of SDS(10%) and 125 μl of proteinase K (20 mg/ml) to the mixture, it wasgently stirred and allowed to stand at 50° C. for 8 hours. This wasfollowed by phenol/chloroform treatment and finally ethanolprecipitation to obtain purified genomic DNA. A 5 μg portion of theobtained genomic DNA was completely digested with the restrictionendonuclease EcoRI (NEB Inc.), and an N-myc probe was used to determineamplification of N-myc by Southern hybridization.

3. Preparation of mRNA from Clinical Tissue of Neuroblastoma withFavorable Prognosis

A 2-3 g portion of the clinical tissue samples of neuroblastoma judgedto have favorable prognosis in 2. above was treated using a Total RNAExtraction Kit (QIAGEN Inc.) and the total RNA was extracted. Theextracted total RNA was purified using an oligo dT cellulose column(Collaborative Research, Inc.) to obtain a pool of mRNA with a polyAstructure.

4. Dephosphorylation of mRNA

A 100-200 μg portion of the mRNA pool prepared in 3. above was dissolvedin 67.3 μl of distilled sterile water containing 0.1% diethylpyrocarbonate (DEPC), and then 20 μl of 5×BAP buffer (Tris-HCl (500 mM,pH=7.0)/mercaptoethanol (50 mM)), 2.7 μl of RNasin (40 unit/μl: PromegaInc.) and 10 μl of BAP (0.25 unit/μl, bacteria-derived alkaliphosphatase: Takara Shuzo Co. Ltd.) were added. The mixture was reactedat 37° C. for 1 hour to effect dephosphorylation of the 5′ end of themRNA. This was followed by phenol/chloroform treatment two times, andfinally ethanol precipitation to obtain a purified dephosphorylated mRNApool.

5. Decapping of Dephosphorylated mRNA

The total amount of the dephosphorylated mRNA pool prepared in 4. abovewas dissolved in 75.3 μl of distilled sterile water containing 0.1%DEPC, and then 20 μl of 5×TAP buffer (sodium acetate (250 mM,pH=5.5)/mercaptoethanol (50 mM), EDTA (5 mM, pH=8.0)), 2.7 μl of RNasin(40 unit/μl) and 2 μl of TAP (tobacco acid pyrophosphatase: 20 unit/μl)were added. The mixture was reacted at 37° C. for 1 hour to effectdecapping treatment of the 5′ end of the dephosphorylated mRNA. Thedephosphorylated mRNA of incomplete length with no capped structureremained without decapping, and with the 5′ end dephosphorylated. Thiswas followed by phenol/chloroform treatment and ethanol precipitation toobtain a purified decapped mRNA pool.

6. Preparation of Oligo-Capped mRNA

The total amount of the decapped mRNA pool prepared in 5. above wasdissolved in 11 μl of distilled sterile water containing 0.1% DEPC, andthen 4 μl of 5′-oligo RNA (5′-AGCAUCGAGUCGGCCUUGGCCUACUGG-3′:100 ng/μl),10 μl of 10× ligation buffer (Tris-HCl (500 mM, pH=7.0)/mercaptoethanol(100 mM)), 10 μl of magnesium chloride (50 mM), 2.5 μl of ATP (24 mM),2.5 μl of RNasin (40 unit/μl), 10 μl of T4 RNA ligase (25 unit/μl:Takara Shuzo Co. Ltd.) and 50 ml of polyethylene glycol (50% w/v,PEG8000: Sigma Corporation) were added. The mixture was reacted at 20°C. for 3 hours for ligation of the 5′-oligo RNA to the 5′ end of thedecapped mRNA. The dephosphorylated mRNA of incomplete length with nocapped structure resulted in no ligation to the 5′-oligo RNA. This wasfollowed by phenol/chloroform treatment and ethanol precipitation toobtain a purified oligo-capped mRNA pool.

7. Removal of DNA from Oligo-Capped mRNA

The oligo-capped mRNA pool prepared in 6. above was dissolved in 70.3 μlof distilled sterile water containing 0.1% DEPC, and then 4 μl ofTris-HCl (1 M, pH=7.0), 5.0 μl of DTT (0.1 M), 16 μl of magnesiumchloride (50 mM), 2.7 g 1 of RNasin (40 unit/μl) and 2 μl of DNaseI (5unit/μl: Takara Shuzo Co. Ltd.) were added. The mixture was reacted at37° C. for 10 minutes to dissolve the excess DNA. This was followed byphenol/chloroform treatment and ethanol precipitation and columnpurification (S-400HR: Pharmacia Biotech Inc.), to obtain a purified DNA(−) oligo-capped mRNA pool.

8. Preparation of 1st Strand cDNA

The DNA (−) oligo-capped mRNA pool prepared in 7. above was reversetranscribed using SuperScript II (kit by Life Tech Oriental, Inc.) toobtain a pool of 1st strand cDNA. The pool of DNA (−) oligo-capped mRNAwas dissolved in 21 μl of sterile distilled water, and then 10 μl of 10×First Strand buffer (kit accessory), 8 μl of dNTP mix (5 mM, kitaccessory), 6 μl of DTT (0.1 M, kit accessory), 2.5 μl of oligo-dTadapter primer (5 pmol/μl,5′-GCGGCTGAAGACGGCCTATGTGGCCTTTTTTTTTTTTTTTTT-3′), 2.0 g 1 of RNasin (40unit/μl) and 2 μl of SuperScript II RTase (kit accessory) were added.The mixture was reacted at 42° C. for 3 hours to effect reversetranscription. This was followed by phenol/chloroform treatment, alkalitreatment and neutralization treatment to dissolve all the RNA andpurification was carried out by ethanol precipitation.

9. Preparation of 2nd Strand cDNA

The 1st strand cDNA pool prepared in 8. above was subjected to PCRamplification using Gene Amp (kit by Perkin Elmer Inc.). The pool of 1ststrand cDNA was dissolved in 52.4 u 1 of sterile distilled water, andthen 30 μl of 3.3× Reaction buffer (kit accessory), 8 μl of dNTP mix(2.5 mM, kit accessory), 4.4 μl of magnesium acetate (25 mM, kitaccessory), 1.6 μl of Primer F (10 pmol/μl,5′-AGCATCGAGTCGGCCTTGTTG-3′), 1.6 μl of Primer R (10 pmol/μl,5′-GCGCTGAAGACGGCCTATGT-3′) and 2 μl of rTth (kit accessory) were added.A 100 μl portion of mineral oil was gently added to the mixture andoverlayed thereon. After denaturing the reaction solution at 94° C. for5 minutes, a cycle of 94° C. for 1 minute, 52° C. for 1 minute and 72°C. for 10 minutes was repeated 12 times, and then the solution wasallowed to stand at 72° C. for 10 minutes to complete the PCR reaction.This was followed by phenol/chloroform treatment and ethanolprecipitation to obtain a 2nd strand cDNA pool.

10. SfiI Treatment of 2nd Strand cDNA

The 2nd strand cDNA pool prepared in 9. above was dissolved in 87 μl ofsterile distilled water, and then 10×NEB buffer (NEB Inc.), 100×BSA(bovine serum albumin available from NEB Inc.) and 2 μl of SfiI(restriction endonuclease, 20 unit/μl, NEB Inc.) were added. The mixturewas reacted overnight at 50° C. to effect SfiI restriction endonucleasetreatment. This was followed by phenol/chloroform treatment and ethanolprecipitation to obtain a pool of cDNA which had been SfiI-treated atboth ends.

11. Size Fractionation of SfiI-Treated cDNA

The SfiI-treated cDNA pool prepared in 10. above was electrophoresed on1% agarose gel and a fraction with >2 kb was purified using Geneclean II(Bio101 Inc.). The purified cDNA pool was dissolved in 100 μl of steriledistilled water and allowed to stand at 37° C. for 6 hours. This wasfollowed by phenol/chloroform treatment and ethanol precipitation toobtain a long-chain cDNA pool.

12. cDNA Library

The long-chain cDNA pool prepared in 11. above was ligated into thecloning vector pME18S-FL3 (provided by Prof. Sumio Kanno of theInstitute of Medical Science, Tokyo University) using a DNA Ligation Kitver.1 (kit by Takara Shuzo Co. Ltd.). The long-chain cDNA pool wasdissolved in 8 μl of sterile distilled water, and then 1 μl ofpME18S-FL3 pretreated with restriction endonuclease DraIII, 80 μl ofSolution A (kit accessory) and 10 μl of Solution B (kit accessory) wereadded and reaction was conducted at 16° C. for 3 hours. This wasfollowed by phenol/chloroform treatment and ethanol precipitation forpurification to obtain a cDNA library.

Example 2 Transformation into E. coli

1. Cloning

The cDNA library prepared in Example 1-12. above was used fortransformation into E. coli (TOP-10: Invitrogen Corporation). The cDNAlibrary was dissolved in 10 μl of sterile distilled water and mixed withTOP-10. The mixture was then incubated on ice for 30 minutes, at 40° C.for 1 minute and on ice for 5 minutes. After adding 500 μl of SOBmedium, shake culturing was performed at 37° C. for 60 minutes.Appropriate amounts thereof were seeded onto ampicillin-containing agarmedia and culturing was continued at 37° C. for a day and a night toobtain E. coli clones.

2. Preservation of E. coli Clones (Preparation of Glycerol Stock)

The E. coli clones on agar media obtained in 1. above were collectedwith toothpick and suspended in 120 μl of LB medium prepared in a96-well plate. The 96-well plate was then allowed to stand overnight at37° C. for culturing of the E. coli. A 72 μl portion of 60% glycerolsolution was then added and preserved at −20° C. (glycerol stock).

Example 2 Sequencing 1. Preparation of Plasmid

The 10 g 1 of glycerol stock prepared in Example 1-2 above wastransferred to a 15 ml centrifugation tube, and then 3 ml of LB mediumand 50 μg/ml of ampicillin were added and shaking was carried outovernight at 37° C. for culturing of the E. coli. A QIAprep SpinMiniprep Kit (QIAGEN Inc.) was then used to extract and purify a plasmidDNA from the E. coli.

2. Analysis of Both End Sequences

Both end sequences of the plasmid DNA prepared in 1. above weredetermined using a DNA Sequencing Kit (kit by ABI). There were combined600 ng of plasmid DNA, 8 μl of premix (kit accessory) and 3.2 pmol ofprimers, and sterile distilled water was added to a total of 20 μl.After denaturing the mixture at 96° C. for 2 minutes, a cycle of 96° C.for 10 seconds, 50° C. for 5 seconds and 60° C. for 4 minutes wasrepeated 25 times for reaction. The product was then purified by ethanolprecipitation. Sequence determination was carried out by polyaqcrylamidegel electrophoresis under denaturing conditions, using ABI377 (ABI).

Example 3 Homology Search of Database

An internet-mediated base sequence homology search was conducted for thebase sequence data obtained from the both end-sequence analysis inExample 2. The search was conducted using the BLAST database of the NCBI(National Center of Biotechnology Information,http://www.ncbi.nblm.nih.gov/BLAST). As a result of the homology search,nbla0078 (one of the cDNA samples) showed high homology to the genomicsequence on human chromosome No. 9 (GeneBank Accession No. AL161625).

Example 4 Cloning of the Full-Length nbla0078

For the genomic sequence obtained in Example 3, its gene transcriptionsequence was deduced using GENESCAN (Burge C et al.: 1997, 1998) andFGENESH (Salamov A A et al.: 1999). Based on the putative sequence thecloning of the full-length of nbla0078 was conducted according to themethod described below.

Specifically, 15 μg of total RNA extracted from a clinical tissue ofneuroblastoma with favorable prognosis was reverse transcribed to cDNAusing superscript II reverse transcriptase (GIBCO). Thereverse-transcribed cDNA (2 μl), 5 μl of sterile distilled water, 1 μlof 10×rTaq buffer (Takara Shuzo Co., Ltd.), 1 μl of 2 mM dNTPs, 0.5 μleach of the synthesized primer set and 0.5 μl of rTaq (Takara Shuzo Co.,Ltd.) were combined. After denaturing the mixture at 95° C. for 2minutes, a cycle of 95° C. for 15 seconds, 58° C. for 15 seconds and 72°C. for 20 seconds was repeated 35 times, and then the mixture wasallowed to stand at 72° C. for 20 minutes for PCR reaction. The bandsamplified by PCR were subcloned into a pGEM-T easy vector (PromegaCorporation) and the base sequences were determined according to astandard method (Sanger F. et al.: Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)). AB1377 (ABI) was used for analysis and both strandsof all the base sequence were analyzed.

The gene sequence of NEDL-1 obtained was registered with DDBJ, GeneBank,EMBL. The accession number was AB048365.

Example 5 Comparison of Gene Expression Levels in Human Neuroblastomaswith Favorable Prognosis and Unfavorable Prognosis by Semi-QuantitativePCR

All semi-quantitative RT-PCR reactions were performed in the mannerdescribed below.

1. Reverse Transcription (RT)

The extracted total RNA (5 μg) was reverse-transcribed into cDNA using aSuperscript II reverse transcriptase (GIBCO).

2. PCR

PCR was performed with rTaq (Takara Shuzo Co., Ltd.). Thereverse-transcribed cDNA (2 μl), 5 μl of sterile distilled water, 1 μlof 10×rTaq buffer, 1 μl of 2 mM dNTPs, 0.5 μl each of the synthesizedprimer set and 0.5 μl of rTaq were combined. After denaturing themixture at 95° C. for 2 minutes, a cycle of 95° C. for 15 seconds, 58°C. for 15 seconds and 72° C. for 20 seconds was repeated 35 times, andthen the mixture was allowed to stand at 72° C. for 20 minutes for PCRreaction.

GAPDH was used as the positive control. Primers are shown below. FW:5′CTGCACCAACAATATCCC3′ (SEQ ID NO:3) RV: 5′GTAGAGACAGGGTTTCAC3′ (SEQ IDNO:4)3. Comparison of NEDL-1 Gene Expression Levels

RT-PCR was performed on the total RNAs of neuroblastomas with favorableprognosis and with unfavorable prognosis obtained in Preparation Example1-3 under the conditions described above. These reaction solutions wereelectrophoresed on 2.5% agarose gel. The results confirmed that theexpression of the NEDL-1 gene was specific for the neuroblastomaclinical tissues with favorable prognosis. Results are shown in FIG. 2.Here, in FIG. 2 the samples in each lane are as follows:

-   Lanes F1-16 (left): neuroblastoma clinical samples with favorable    prognosis-   Lanes UFl-16 (right): neuroblastoma clinical samples with    unfavorable prognosis-   Control: GAPDH-   Positive control (favorable prognosis): TrkA-   Negative control (unfavorable prognosis): NMYC

Example 6 Tissue-Dependent Gene Expression Levels by Semi-QuantitativePCR

mRNAs of normal human tissues (Clontech) were used to perform RT-PCRunder the conditions described in Example 5. These reaction solutionswere electrophoresed on 2.5% agarose gel. The results confirmed that theexpression of the NEDL-1 gene expression was tissue-specific among thenormal human tissues. Results are shown in FIG. 3. The expression ofNEDL-1 was restricted in the brain, the fetal brain, the cerebellum andthe kidney.

Example 7 Gene Expression Levels that are Dependent on NeuroblastomaCell Lines by Semi-Quantitative PCR

RT-PCR was performed on the total RNAs of various neuroblastoma celllines under the conditions described in Example 5. These reactionsolutions were electrophoresed on 2.5% agarose gel. The resultsconfirmed that the distribution of NEDL-1 gene expression wastissue-specific. Results are shown in FIG. 3B. Those with which NEDL-1expression was observed were SKN-DZ, TGW, KAN, KCN+8, and LAN-5.

Example 8 Northern Hybridization

A multi tissue Northern blot on which poly(A) ⁺RNA of different humantissues had been blotted was used together with NEDL-1 cDNA (labeledwith ³²p) as a probe to carry out hybridization. A β-actin cDNA probewas used as control. Results are shown in FIG. 4. Two transcripts withabout 10.0 kb and about 7.0 kb were observed in the brain, the kidneyand the fetal brain.

Example 9 Ubiquitin Ligase Activity

Equivalent amounts of a bacterial lysis product expressing E2 (UbCH5c orUbCH7) were incubated with ubiquitin, yeast E1 and E3 (Nedd4, NEDL-1 orNEDL-2) at 37° C. for 2 hours. Subsequently, the product was separatedon SDS-PAGE under reductive conditions and blotted with anti-ubiquitinantibody. Purified recombinant GST-Nedd1, GST-NEDL-1/HECT andGST-NEDL2/HECT were respectively used as E3 (ubiquitin ligase). Resultsare shown in FIG. 5. Ubiquitination increased depending on the amount ofE3 (regions enclosed by dotted line in the figure). NEDL-1 displayedubiquitin ligase activity at the same level as Nedd4 which served aspositive control.

Example 10 Cellular Localization of NEDL-1

The full-length NEDL-1 gene was transfected into Cos 7 cellstransiently. Forty eight hours later, the cells were lysed, subjected toSDS-PAGE on 6% polyaqcrylamide and analyzed with NEDL-1 antibody. Eachgene product was detected at the position of about 220 kD. Results areshown in FIG. 6A. In endogenous expression (CHP134 cells) and exogenousexpression (Cos 7 cells) NEDL-1 was mainly localized in the cytoplasmand the cell membrane. The result is well in accord with those from theother members of the Nedd4 family (FIG. 6B).

Example 11 Interaction Between NED-1 and AICD

A typical yeast two-hybrid screening was performed using a MATCHMAKERGAL4 Two-HYBRID SYSTEM2 (K1604-1: Clontech Company) with the NEDL-1 WWdomain region as a DNA binding domain fusion protein. Specifically, PCRcloning was carried out in pAS2-1 (GenBank Accession No. U3-4907) inframe and sequencing was carried out with a DNA sequencer ABI PRISM 377(Perkin Elmer/Applied Biosystems). CG-1945 cell line was used as adirecting yeast cell line and a Human fetal Brain MATCHMAKER cDNALibrary (Priming Method: Xho I-(dT)15/Vector: pACT2/Cat. #HL4028AH) wasused as a library. Proliferation potency was assayed in a SD (-His,-Trp) TPD plate and library screening was performed in a YPD medium withthe addition of 3-amino-1,2,4-triazole (20 mM) which was an inhibitor ofHIS3. HIS+colonies were picked up and assayed for their β-galactosidaseactivity according to a standard method, where positive clones wereselected. Plasmid DNAs were collected from these positive clonesaccording to a standard method. Following the above procedure, a clonehaving the AICD region was found in a plurality of positive clones thathad been discovered by performing the yeast two-hybrid screening. Theclone was focused and investigation proceeded.

A FLAG sequence (DYKDDDDK) was appended to the ACID region of the cloneand a mammalian cell expression vector capable of expression under a CMVpromoter was constructed. After sequence confirmation with the sequencerdescribed above, the vector was transiently coexpressed with aCMV-NEDL-1 expression vector in the cells. Thus investigation wascarried out to see if the physical interaction could be reproduced in acell.

Cos7 cells were maintained at 80% confluency in a Dulbecco's modifiedEagle's medium containing 10% FBS. Each 6 μg of DNA was used to carryout transient gene transfer according to the Lipofection method.

LipofectAMINE plus (Life technologies, Inc.) was used as a liposomeagent. Forty eight hours later cells were washed twice with PBS on iceand 1 ml of TNEBuffer (10 mM Tris-HCl, pH 7.8/1% NP40/0.15 M NaCl/1 mmEDTA/10 μl aprotinin) was added thereto. Incubation was carried out onice for 10 minutes. The cells were then transferred to an Eppendorf tubeand after addition of 20 μl of Protein B-Sepharose (50% slurry), thecells were revolved at 4° C. for 30 minutes to eliminate non-specificbonding. Then cells were centrifuged at 15,000 rpm/30 minutes at 4° C.The supernatant was transferred to a new Eppendorf tube by decantationand after addition of 30 μl of Protein B-Sepharose and 10 μl of anti-NED1 antibody, the cells were revolved at 4° C. for 3 hours. The cells werethen spanned down and were washed with THE Buffer four times.Subsequently, 25 μl of TNE buffer and 25 μl of ×2 sample buffer wereadded to the cells and the mixture was boiled for 5 minutes to preparesamples for electrophoresis. The same amounts of protein were developedon Tricine SDS-PAGE using 15% aqcrylamide gel, transcribed onto a PVDFmembrane and blocked with 3% BSA. After antibody reaction withanti-FLAG-M2 antibody (Sigma) as a primary antibody and with anti-mouseIgG antibody labeled with HRP as a secondary antibody, the proteins weredetected with an ECL Western Blotting Detection Reagent (code no.RPN2106).

FIG. 7 shows the immunoprecipitation with anti-NEDL-1 antibody followedby the detection with anti-FLAG antibody. Lanes 2 and 3 and Lanes 6 and7 are a set; Lanes 4 and 5 and Lanes 8 and 9 are a set. The respectivesets resulted from two different, independent clones. FLAG-ACIDcoprecipitated with NEDL-1 (Lanes 2 and 3). The FLAG-AICD protein bandexpressed is weak in a normal Western Blotting and this is due to theinstability of the protein (Lanes 4 and 5). Lanes 4 and 5/8 and 9 arenegative controls. According to the normal Western Blotting strongexpression was observed in Lanes 8 and 9, while coprecipitation withNEDL-1 was not observed in Lanes 4 and 5. FIG. 8 shows theimmunoprecipitation with anti-FLAG antibody followed by the detectionwith anti-NEDL-antibody. Only where NEDL-1 and FLAG-ACID wereco-existed, strong coprecipitation band was obtained (Lanes 4 and 5),which shows direct interaction of the two.

Example 12 Ubiquitination of BAPP and ACID by NEDL-1

Yeast two-hybrid screening as described in Example 1 was performed,except that prior to harvesting treatment with MG132 (20 μM), aproteasome inhibitor, was done for 2 hours. The other experimentalprocedures followed almost Example 11. FIGS. 9 and 10 show the resultsof immunoprecipitation.

FIG. 9A shows the immunoprecipitation with anti-HA antibody followed byblotting with anti-ubiquitin antibody. It can be seen that the absoluteamount of the ubiquitinated molecules in the cell has increased in thepresence of NEDL-1. FIG. 10 shows the immunoprecipitation with anti-FLAGantibody followed by blotting with anti-ubiquitin antibody. FIG. 9Bshows the immunoprecipitation with anti-HA antibody followed by blottingwith an antibody recognizing ACID. A high molecular weight smear bandwas observed upward starting from βAPP. This suggests the ubiquitinationof βAPP. From FIGS. 9A and 9B it is obvious that βAPP has been subjectedto ubiquitination in the presence of NEDL-1. However, its degree is notrelated to the mutation of βAPP (whether WT or MT) (Lanes 1 and 2, andLanes 3 and 4 both in FIG. 9A and FIG. 9B). Lanes 5 and 6 in FIG. 9A andLanes 7 and 8 in FIG. 10 show the ubiquitination of AICD. FLG-ACID has amolecular weight of about 7 KD (FIG. 9B). Anti-ubiquitin antibody wasused for detection in FIG. 9A and FIG. 10, where FLG-ACID that was notubiquitinated does not appear (lowest column). As a ubiquitin moleculewith about 9 kD adds to FLAG-AICD, bands increasing by the about 9 kDare detected with anti-ubiquitin antibody (indicated as asterisks in thefigure). It is understood that ACID alone is subjected to ubiquitinationby NEDL-1. In the figure, bands that look two-lines represent thedifferential molecular weight between the exogenous ubiquitin with a HAtag added and the endogenous ubiquitin with no tag.

Example 13 Interaction Between NEDL-1 and SOD1 Mutants

The yeast two-hybrid screening as described in Example 11 was performedon SOD1 genes. The experimental procedure generally followed Example 11.Specifically, SOD1 genes (wild type and mutant types) were transientlycoexpressed with CMV-NEDL-1 in cells.

After immunoprecipitation with anti-NEDL-1 antibody, the cells werewashed to prepare samples for electrophoresis. The same amounts ofprotein were developed on 15% SDS-PAGE, transcribed onto a PVDF membraneand allowed for antibody reaction with anti-FLAG antibody, after whichdetection by ECL was carried out. Results are shown in FIG. 11.Similarly, after immunoprecipitation with anti-FLAG antibody, the cellswere washed to prepare samples for electrophoresis. The same amounts ofprotein were developed on 6% SDS-PAGE, transcribed onto a PVDF membraneand allowed for antibody reaction with anti-NEDL-1 antibody, after whichdetection by ECL was carried out. Results are shown in FIG. 12. In Lane3 NEDL-1 and SOD1(WT) did not coprecipitate. In Lanes 4 and 5 SOD1(A4V)and SOD1(C6F) strongly interacted with NEDL-1 and coprecipitated:SOD1(A4V) and SOD1(C6F) were mutants that caused rapid clinical progressafter crisis and death within one year. In Lane 6 SOD1 (H46R) showedvery week interaction with NEDL-1: SOD1(H46R) displayed slow clinicalprogress after crisis and made survival of nearly 40 years possible. InLane 7 SOD1(G93A) showed medium interaction with NEDL-1: SOD1(G93A) wasa mutant that displayed s peculiar neural symptom after crisis.

The test results shown in FIGS. 11 and 12 have revealed that NEDL-1 doesnot interact with SOD1 of the wild type but interacts with the SOD1mutants which are responsible for familial ALS. Further, it has beenfound that the degree of interaction roughly correlates with the degreeof clinical malignancy.

Example 14 Ubiquitination of SOD1 Mutants by NEDL-1

The yeast two-hybrid screening as described in Example 1 was performed,except that prior to harvesting treatment with MG132 (20 μM), aproteasome inhibitor, was done for 2 hours. The other experimentalprocedures almost followed Example 11. FIG. 13 show the results ofimmunoprecipitation, where the products were immunoprecipitated withFLAG and then blotted with anti-ubiquitin antibody. The SOD1 mutantswere ubiquitinated in the absence of NEDL-1. See Lanes 1, 3, 5 and 7 inFIG. 13. The degree of ubiquitination is 3>5>7>1; it correlates with theclinical severity of the mutants (as explained above). This suggests thepossibility that a ubiquitin ligase (such as dorfin) existing in a cellto manage quality control is involved. (Niwa J., Ishigaki S., Doyu M.,Suzuki T., Tanaka K., Sobue G. A., Novel centrosomal ring-fingerprotein, dorfin, mediates ubiquitin ligase activity. Biochem. Biophys.Res Commun. 2001 Mar. 2; 281 (3): 706-13.)

It is also understood that the degree of ubiquitination drasticallyincreases in the presence of NEDL-1. See Lanes 2, 4, 6, and 8 in FIG.13. The ubiquitination degree is again 4>6>8>2 and correlates with theclinical severity of the mutants. It is thus understood that NEDL-1 hasthe function, as ubiquitin ligase of the quality control type, ofstrongly exhibiting the ubiquitination power against SOD1 mutants asopposed to mutant BAPP.

INDUSTRIAL APPLICABILITY

As described above, the nucleic acid probes and primers according tothis invention may be used for various types of hybridization or PCR,and permit detection of the expression of the NEDL-1 gene in not onlyneuroblastomas but also other human tissues and cells, as well as theanalysis of its structure and function. Production of the NEDL-1 proteinencoded by the gene through genetic engineering is also possibleaccording to the invention. The protein has been confirmed for itsubiquitin ligase activity and has been shown to be a ubiquitin ligase ofthe HECT type based on its structure. Accordingly, the identification ofthe substrate for the NEDL-1 protein in the ubiquitin-proteasome systemhas been possible and it will lead to the possibility of treatingneurodegenerative diseases involving the protein. In reality, it wasdetermined that NEDL-1 interacted with β APP, AICD or SOD1 (mutanttype). Further, this interaction was identified to be throughubiquitination.

The nucleic acids according to this invention are those derived from theNEDL-1 gene whose expression is enhanced in neuroblastoma with favorableprognosis, and therefore allow the diagnosis for the prognosis ofneuroblastoma based on this genetic information from these nucleicacids. Unlike the N-myc gene which is a factor for unfavorableprognosis, these genes are considered factors for favorable prognosis,similarly to the TrkA gene, and therefore can serve as markers (tumormarkers) for neuroblastoma malignancy and sensitivity to anti-canceragents. Specifically, the nucleic acid probes of this invention or theprimers of the invention may be used to construct the diagnostic agentsor diagnostic kits for the prognosis of neuroblastoma and to detect theNEDL-1 protein or the NEDL-1 protein in clinical tissue samples, wherebythe prognosis can be diagnosed.

1. A nucleic acid probe comprising nucleic acid (a) or nucleic acid (b):(a) a nucleic acid having a portion of a base sequence set forth in SEQID NO:2 in the Sequence Listing or a base sequence complementarythereto; and (b) a nucleic acid capable of hybridizing to the nucleicacid comprising a base sequence set forth in SEQ ID NO:2 in the SequenceListing, or having a base sequence complementary to said base sequence.2. The nucleic acid probe according to claim 1, wherein the nucleic acidis DNA.
 3. The nucleic acid probe according to claim 1 or 2, wherein thenucleic acid has a base length of at least 20 bases.
 4. The nucleic acidprobe according to claim 3, wherein the base sequence set forth in SEQID NO:2 is a full-length thereof.
 5. A diagnostic agent for theprognosis of neuroblastoma, the agent comprising the nucleic acid probeaccording to any of claims 1-4, as the effective ingredient.
 6. A primercontaining DNA (a) or DNA (b): (a) DNA having a portion of a basesequence set forth in SEQ ID NO:2 in the Sequence Listing or a basesequence complementary thereto; or (b) DNA capable of hybridizing to theDNA comprising a base sequence set forth in SEQ ID NO:2 in the SequenceListing, or having a base sequence complementary to said base sequence.7. A kit for diagnosing the prognosis of neuroblastoma, the kitcomprising the primer according to claim 6 as the effective ingredient.8. A method for diagnosing the prognosis of neuroblastoma, the methodcomprising detecting the presence or absence of a nucleic acidcomprising a base sequence set forth in SEQ ID NO:2 in the SequenceListing in a clinical tissue sample of neuroblastoma.
 9. A method fordiagnosing the prognosis of neuroblastoma, the method comprisingdetecting the presence or absence of a protein comprising an amino acidsequence set forth in SEQ ID NO:1 in the Sequence Listing in a clinicaltissue sample of neuroblastoma.
 10. A method for diagnosing theprognosis of neuroblastoma, the method comprising contacting with aclinical tissue sample of neuroblastoma, (a) a nucleic acid having aportion of a base sequence set forth in SEQ ID NO:2 in the SequenceListing or a base sequence complementary thereto or (b) a nucleic acidcapable of hybridizing to the nucleic acid comprising a base sequenceset forth in SEQ ID NO:2 in the Sequence Listing, or having a basesequence complementary to said base sequence; and analyzing theexpression of a protein comprising an amino acid sequence set forth inSEQ ID NO:1 in the Sequence Listing or an level thereof.
 11. Apolyubiqutination agent comprising as the effective ingredient, aprotein comprising a base sequence set forth in SEQ ID NO: 1 in theSequence Listing.
 12. The polyubiqutination agent according to claim 11,wherein the substrate to be ubiquitinated is β-amyloid precursor protein(βAPP).
 13. The polyubiqutination agent according to claim 11, whereinthe substrate to be ubiquitinated is β-amyloid precursor proteinintracellular region (AICD)
 14. The polyubiqutination agent according toclaim 11, wherein the substrate to be ubiquitinated is a superoxidedismutase mutant (SOD1).
 15. A composition for modulating β-amyloidprecursor protein (APP), the composition comprising an effective amountof a protein comprising an amino acid sequence set forth in SEQ ID NO:1in the Sequence Listing to modulate the expression, the production orthe formation of β-amyloid precursor protein in a cell.
 16. Acomposition for modulating β-amyloid precursor protein (βAPP), thecomposition comprising an effective amount of a nucleic acid comprisinga base sequence set forth in SEQ ID NO:2 in the Sequence Listing tomodulate the expression, the production or the formation of β-amyloidprecursor protein in a cell.
 17. A method for modulating the expression,the production or the formation of β-amyloid precursor protein in acell, the method comprising administering an effective amount of aprotein comprising an amino acid sequence set forth in SEQ ID NO:1 inthe Sequence Listing to modulate the expression, the production or theformation of β-amyloid precursor protein in a cell.
 18. A method formodulating the expression, the production or the formation of β-amyloidprecursor protein in a cell, the method comprising administering aneffective amount of a nucleic acid comprising a base sequence set forthin SEQ ID NO:2 in the Sequence Listing to modulate the expression, theproduction or the formation of β-amyloid precursor protein in a cell.19. A composition for modulating superoxide dismutase (SOD1) activity,the composition comprising an effective amount of a protein comprisingan amino acid sequence set forth in SEQ ID NO:1 in the Sequence Listingto modulate the superoxide dismutase (SOD1) activity in a cell.
 20. Acomposition for modulating superoxide dismutase (SOD1) activity, thecomposition comprising an effective amount of a nucleic acid comprisinga base sequence set forth in SEQ ID NO:1 in the Sequence Listing tomodulate the superoxide dismutase (SOD1) activity in a cell.
 21. Thecomposition for modulating superoxide dismutase (SOD1) activityaccording to claim 19 or 20, wherein the superoxide dismutase (SOD1) isa mutant type.
 22. A method for modulating superoxide dismutase activityin a cell, the method comprising administering to the cell, an effectiveamount of a protein comprising an amino acid sequence set forth in SEQID NO:1 in the Sequence Listing to modulate the superoxide dismutase(SOD1) activity.
 23. A method for modulating superoxide dismutase (SOD1)activity in a cell, the method comprising administering to the cell, aneffective amount of a nucleic acid comprising a base sequence set forthin SEQ ID NO:2 in the Sequence Listing to modulate the superoxidedismutase (SOD1) activity.
 24. The method according to claim 22 or 23,wherein the superoxide dismutase (SOD1) is a mutant type.